Fate mapping melanoma persister cells through regression and into recurrent disease in adult zebrafish.

Melanoma heterogeneity and plasticity underlie therapy resistance. Some tumour cells possess innate resistance, while others reprogramme during drug exposure and survive to form persister cells, a source of potential cancer cells for recurrent disease. Tracing individual melanoma cell populations through tumour regression and into recurrent disease remains largely unexplored, in part, because complex animal models are required for live imaging of cell populations over time. Here, we applied tamoxifen-inducible creERt2/loxP lineage tracing to a zebrafish model of MITF-dependent melanoma regression and recurrence to image and trace cell populations in vivo through disease stages. Using this strategy, we show that melanoma persister cells at the minimal residual disease site originate from the primary tumour. Next, we fate mapped rare MITF-independent persister cells and demonstrate that these cells directly contribute to progressive disease. Multiplex immunohistochemistry confirmed that MITF-independent persister cells give rise to Mitfa+ cells in recurrent disease. Taken together, our work reveals a direct contribution of persister cell populations to recurrent disease, and provides a resource for lineage-tracing methodology in adult zebrafish cancer models.

Assaying sensory ciliopathies using calcium biosensor expression in zebrafish ciliated olfactory neurons.

BACKGROUND: Primary cilia mediate signal transduction by acting as an organizing scaffold for receptors, signalling proteins and ion channels. Ciliated olfactory sensory neurons (OSNs) organize olfactory receptors and ion channels on cilia and generate a calcium influx as a primary signal in odourant detection. In the zebrafish olfactory placode, ciliated OSNs and microvillus OSNs constitute the major OSN cell types with distinct odourant sensitivity. METHODS: Using transgenic expression of the calcium biosensor GCaMP5 in OSNs, we analysed sensory cilia-dependent odour responses in live zebrafish, at individual cell resolution. oval/ift88 mutant and ift172 knockdown zebrafish were compared with wild-type siblings to establish ciliated OSN sensitivity to different classes of odourants. RESULTS: oval/ift88 mutant and ift172 knockdown zebrafish showed fewer and severely shortened OSN cilia without a reduction in OSN number. The fraction of responding OSNs and response amplitudes to bile acids and food odour, both sensed by ciliated OSNs, were significantly reduced in ift88 mutants and ift172-deficient embryos, while the amino acids responses were not significantly changed. CONCLUSIONS: Our approach presents a quantitative model for studying sensory cilia signalling using zebrafish OSNs. Our results also implicate ift172-deficiency as a novel cause of hyposmia, a reduced sense of smell, highlighting the value of directly assaying sensory cilia signalling in vivo and supporting the idea that hyposmia can be used as a diagnostic indicator of ciliopathies.

Wilms Tumor 1b defines a wound-specific sheath cell subpopulation associated with notochord repair.

Regenerative therapy for degenerative spine disorders requires the identification of cells that can slow down and possibly reverse degenerative processes. Here, we identify an unanticipated wound-specific notochord sheath cell subpopulation that expresses Wilms Tumor (WT) 1b following injury in zebrafish. We show that localized damage leads to Wt1b expression in sheath cells, and that wt1b+cells migrate into the wound to form a stopper-like structure, likely to maintain structural integrity. Wt1b+sheath cells are distinct in expressing cartilage and vacuolar genes, and in repressing a Wt1b-p53 transcriptional programme. At the wound, wt1b+and entpd5+ cells constitute separate, tightly-associated subpopulations. Surprisingly, wt1b expression at the site of injury is maintained even into adult stages in developing vertebrae, which form in an untypical manner via a cartilage intermediate. Given that notochord cells are retained in adult intervertebral discs, the identification of novel subpopulations may have important implications for regenerative spine disorder treatments.

Notochord Injury Assays that Stimulate Transcriptional Responses in Zebrafish Larvae.

Zebrafish have become an increasingly important model organism in the field of wound healing and regenerative medicine, due to their high regenerative capacity coupled with high-resolution imaging in living animals. In a recent study, we described multiple physical and chemical methods to induce notochord injury that led to highly specific transcriptional responses in notochord cellular subpopulations. The notochord is a critical embryonic structure that functions to shape and pattern the vertebrae and spinal column. Here, we describe precision needle injury, tail-notochord amputation, and chemical inhibition of caveolin that trigger a wound-specific wt1b expression response in the notochord sheath cell subpopulation. We propose that these procedures can be used to study distinct cell populations that make up the cellular processes of notochord repair.

Methods for studying the zebrafish brain: past, present and future.

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience.

Dopamine from the brain promotes spinal motor neuron generation during development and adult regeneration.

Coordinated development of brain stem and spinal target neurons is pivotal for the emergence of a precisely functioning locomotor system. Signals that match the development of these far-apart regions of the central nervous system may be redeployed during spinal cord regeneration. Here we show that descending dopaminergic projections from the brain promote motor neuron generation at the expense of V2 interneurons in the developing zebrafish spinal cord by activating the D4a receptor, which acts on the hedgehog pathway. Inhibiting this essential signal during early neurogenesis leads to a long-lasting reduction of motor neuron numbers and impaired motor responses of free-swimming larvae. Importantly, during successful spinal cord regeneration in adult zebrafish, endogenous dopamine promotes generation of spinal motor neurons, and dopamine agonists augment this process. Hence, we describe a supraspinal control mechanism for the development and regeneration of specific spinal cell types that uses dopamine as a signal.

Neural circuits mediating olfactory-driven behavior in fish.

The fish olfactory system processes odor signals and mediates behaviors that are crucial for survival such as foraging, courtship, and alarm response. Although the upstream olfactory brain areas (olfactory epithelium and olfactory bulb) are well-studied, less is known about their target brain areas and the role they play in generating odor-driven behaviors. Here we review a broad range of literature on the anatomy, physiology, and behavioral output of the olfactory system and its target areas in a wide range of teleost fish. Additionally, we discuss how applying recent technological advancements to the zebrafish (Danio rerio) could help in understanding the function of these target areas. We hope to provide a framework for elucidating the neural circuit computations underlying the odor-driven behaviors in this small, transparent, and genetically amenable vertebrate.

Analysis of the astray/robo2 zebrafish mutant reveals that degenerating tracts do not provide strong guidance cues for regenerating optic axons.

During formation of the optic projection in astray/robo2 mutant zebrafish, optic axons exhibit rostrocaudal pathfinding errors, ectopic midline crossing and increased terminal arbor size. Here we show that these errors persist into adulthood, even when robo2 function is conditionally reduced only during initial formation of the optic projection. Adult errors include massive ectopic optic tracts in the telencephalon. During optic nerve regeneration in astray/robo2 animals, these tracts are not repopulated and ectopic midline crossing is reduced compared with unlesioned mutants. This is despite a comparable macrophage/microglial response and upregulation of contactin1a in oligodendrocytes of entopic and ectopic tracts. However, other errors, such as expanded termination areas and ectopic growth into the tectum, were frequently recommitted by regenerating optic axons. Retinal ganglion cells with regenerating axons reexpress robo2 and expression of slit ligands is maintained in some areas of the adult optic pathway. However, slit expression is reduced rostral and caudal to the chiasm, compared with development and ubiquitous overexpression of Slit2 did not elicit major pathfinding phenotypes. This shows that (1) there is not an efficient correction mechanism for large-scale pathfinding errors of optic axons during development; (2) degenerating tracts do not provide a strong guidance cue for regenerating optic axons in the adult CNS, unlike the PNS; and (3) robo2 is less important for pathfinding of optic axons during regeneration than during development.

Sonic hedgehog is a polarized signal for motor neuron regeneration in adult zebrafish.

In contrast to mammals, the spinal cord of adult zebrafish has the capacity to reinitiate generation of motor neurons after a lesion. Here we show that genes involved in motor neuron development, i.e., the ventral morphogen sonic hedgehog a (shha), as well as the transcription factors nkx6.1 and pax6, together with a Tg(olig2:egfp) transgene, are expressed in the unlesioned spinal cord of adult zebrafish. Expression is found in ependymoradial glial cells lining the central canal in ventrodorsal positions that match expression domains of these genes in the developing neural tube. Specifically, Tg(olig2:egfp)(+) ependymoradial glial cells, the adult motor neuron progenitors (pMNs), coexpress Nkx6.1 and Pax6, thus defining an adult pMN-like zone. shha is expressed in distinct ventral ependymoradial glial cells. After a lesion, expression of all these genes is strongly increased, while relative spatial expression domains are maintained. In addition, expression of the hedgehog (hh) receptors patched1 and smoothened becomes detectable in ependymoradial glial cells including those of the pMN-like zone. Cyclopamine-induced knock down of hh signaling significantly reduces ventricular proliferation and motor neuron regeneration. Expression of indicator genes for the FGF and retinoic acid signaling pathways was also increased in the lesioned spinal cord. This suggests that a subclass of ependymoradial glial cells retain their identity as motor neuron progenitors into adulthood and are capable of reacting to a sonic hedgehog signal and potentially other developmental signals with motor neuron regeneration after a spinal lesion.